Artificial Protein Cage with Unusual Geometry and Regularly Embedded Gold Nanoparticles

Artificial protein cages have great potential in a number of areas including cargo capture and delivery and as artificial vaccines. Here, we investigate an artificial protein cage whose assembly is triggered by gold nanoparticles. Using biochemical and biophysical methods we were able to determine both the mechanical properties and the gross compositional features of the cage which, combined with mathematical models and biophysical data, allowed the structure of the cage to be predicted. The accuracy of the overall geometrical prediction was confirmed by the cryo-EM structure determined to sub-5 Å resolution. This showed the cage to be nonregular but similar to a dodecahedron, being constructed from 12 11-membered rings. Surprisingly, the structure revealed that the cage also contained a single, small gold nanoparticle at each 3-fold axis meaning that each cage acts as a synthetic framework for regular arrangement of 20 gold nanoparticles in a three-dimensional lattice.


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Formation of large TRAP-cage (TRAP-LC GNP ) was carried out in similar fashion, but the reaction consisted of equimolar amounts of TRAP(K35C/R64S) (final concentration 1 mM of monomeric subunits) and GNPs in cage buffer. SEC purification typically resulted in large cage eluting with a peak maximum at around 12.0 -13.0 ml.
Cage stability. Final concentration of protein for stability tests was around 0.1 mg/ml. Purified cage stock was prepared in cage buffer for all the tests and mixed with tested agents, ensuring that the volume of the cage stock constituted less than 10% of the total volume. After each test was finished, samples were centrifuged and supernatant containing ~1 µg of protein was mixed with 4X native PAGE sample buffer. Results were monitored by dark blue native PAGE. Supernatant of selected samples was also analysed by TEM. Each test was repeated at least once with similar results.
For thermal tests, each sample was incubated for 10 min. in temperatures between 45-90°C in cage buffer. pH stability tests were performed by diluting cage stock with suitable buffers and incubating overnight at room temperature. Buffers used: 50 mM Gly-HCl, pH 2 or 3; 50 mM sodium acetate, pH 4; 50 mM potassium phosphate, pH 6 or 12; 50 mM Gly-NaOH, pH 10 or 11; 50 mM KCl-NaOH, pH 13.
Tested agents were diluted to suitable concentrations with the same buffer, mixed with cage stock and incubated overnight at room temperature.
Cage disassembly was tested with oxidised (GSSG) and reduced (GSH) forms of glutathione, DTT, TCEP, L-Cys and 3-(diphenylphosphino)benzenesulfonic acid sodium salt ( Transmission electron microscopy (TEM). TEM grids (Formvar/Carbon film-Cu 300 mesh, FC300Cu100, EMResolutions) were glow-discharged in grid mode for 70 s, 8 mA (Leica EM ACE200). Samples for TEM imaging were typically diluted to a final protein concentration of 0.10 mg/ml, 3 μl applied onto prepared grids, negatively stained with 2 μl freshly prepared 3% phosphotungstic acid, pH 7-8, and visualized using a JEOL JEM-1230 80 kV instrument. Cage size measurements and statistics were carried out using ImageJ software. contact mode with a tip speed of 500 nm/s, and an indentation force of 1 nN. Each indented particle was imaged before and after indentation.

Atomic force microscopy (AFM) based nanoindentation. AFM based nanoindentation
The acquired images and force-distance (F-D) curves were processed using JPK data processing software. The determination of the particle spring constant was performed using IgorPro software by following the protocol as described in reference. 2 The F-D curves extracted from indenting the particle shows linear behaviour. Therefore, we used Hooke's law considering two springs in series (particle and cantilever) in order to extract the particle spring constant. Reported errors are standard deviation. Using the averaged dimensional parameters (diameter and thickness) of the particles (both TRAP-SC GNP and TRAP-LC GNP ) and the particle spring constant, the Young's modulus was determined by thin cell theory. 2 For this calculation the thickness of the shell was taken and as radius, the outer radius minus half the thickness.

High Speed-AFM (HS-AFM).
The major challenge in the structural analysis of TRAP-cages using AFM is its structural diversity within a very small particle surface area (particle diameter of 15-22 nm). Using HS-AFM images at high throughput were recorded, with high signal to noise ratio, and at minimal imaging force (<100 pN). HS-AFM (RIBM, Japan) was used in amplitude modulation tapping mode. USC-F1.2-k0.15 cantilevers (Nanoworld) with a nominal spring constant of 0.15 N/m were used. 4,5 The acquired images were processed using Kodec (Kanazawa University, Japan), and ImageJ software. Unless otherwise mentioned, the images were processed minimally (i.e. baseline correction, contrast check etc.).

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The estimation of dihedral angle between the rings was calculated using published procedures. 6 In brief, in reference to Figure S5, the calculation steps are as follows: Step 1 contains the measurements of corrected distances (in x, y and z direction) between two consecutive points. The X, Y, and Z position of the observable ring center (on a cage) was recorded from the images, and distances between two points were calculated (xmeasured and ymeasured). The corresponding measured distances were corrected for tip convolution as follows: Where n is the number of measurements in X and Y direction for each particle. r(z)measured, r(x)i(measured), and r(y)i(measured) are the measurements of the height, x diameter, and y diameter of the corresponding particles, respectively.
In step 2, the calculation of the actual distances (D) between the two ring centers are estimated as follows: In step 3, the dihedral angle between the ring is calculated as follows: (4)

Size Exclusion Chromatography with Right-Angle (RALS)/Low-Angle (LALS) Light
Scattering and Refractive Index (RI) detection. Molecular   with the planarity weight set to three orders of magnitude larger than for the lengths and the angles.
This gave us structures with preserved planarity (with zero planarity distortions modulo numerical error). The energy functional was minimized using a Monte Carlo method and the program output a file containing the vertex coordinates, the topology of the cage, as well as the order of deformations obtained for the angles and edge lengths. The exact explanation of the algorithm will be given elsewhere but some details were already covered in. 7 From the polyhedral cages generated by the method described above, we selected the only candidate for which the dihedral angles and diameter were in line with the AFM measurements, and the deformation level was small (i.e. less than 2%). The modelled cage in Figure 3 suggests a set of 48 S-Au-S bonds, one for each pair of joined hendecagonal vertices.
A small cage model with TRAP ring structures inserted into predicted polyhedron faces was built using Cage Builder tool of UCSF Chimera (version 1.14). 8

Cryogenic electron microscopy (cryo-EM). Cryo-EM structure reconstruction was performed
as described in Figure S7. Raw micrographs were motion corrected on the fly using Warp. 9 Motion corrected micrographs were imported to cryoSPARC and underwent further analysis. CTF estimation was performed by patchCTF job in cryoSPARC v2.15.0. 10 Examples of the statistics of the analyzed micrographs are shown in Figure S8. After CTF estimation, 1,000 particles were manually picked and classified in reference-free 2D classification in order to create templates for the automatic picking job (Figure S7c-d). Template-based picking gave 1,315,498 particles, which were again 2D classified and cleaned from bad particles. As a result, five 2D classes were selected with 671,845 particles Figure S7f). First, ab-initio reconstruction followed by homogenous refinement gave an initial 3D model with 5.93 Å resolution. This model was used to produced new, "artificial" 2D classes by back projection. 50 such classes were produced ( Figure S7g) and used for a second round of template-based particle picking (2,006,399 particles were picked).